Hepatitis C virus (HCV) infects over 1.5% of the world population. It causes hepatitis and cirrhosis and is highly associated with hepato- cellular carcinoma. We propose to identify compounds that inhibit HCV helicase, which is thought to be important for HCV RNA and protein synthesis. Our strategy includes screening of compounds and fungal extracts to identify helicase inhibitors and verification of the antiviral activity in various HCV replication models. To date, we have demonstrated the helicase activity of a recombinant HCV NS3 protein in a scintillation proximity assay (SPA) and formatted and validated the assay for high throughput screening. In phase I, we will complete screening of 100,000 entities (50,000 compounds and 50,000 fungal extracts). Hits, defined as compounds and extracts that inhibit the SPA signal in high throughput screen, will be confirmed by re-screening. In addition, hits that are intrinsic inhibitors of the SPA signal will be identified and excluded. Furthermore, selective hits that inhibit the activity of HCV helicase but not a mammalian helicase will be identified as selective hits. In phase II, we will prioitize the hits to identify nontoxic, selective helicase inhibitors that inhibit HCV replication in a chimpanzee hepatocyte model and mouse model. PROPOSED COMMERCIAL APPLICATION: HCV infects over 1.5% of the world and US population and poses a major health problem since there is no effective treatment or prevention available. Compounds and natural products that inhibit HCV helicase can be developed into novel anti-HCV drugs with enormous commercial potential.